Methods of preserving functionality of an organ, preserving fertility of a patient undergoing a treatment expected to cause sterility and assuring a supply of viable gametes for future use

ABSTRACT

A method of preserving functionality of an organ. The method includes removing a whole organ and associated vasculature, cryo-preserving the whole organ, allowing a period of time to elapse, thawing the whole organ including vasculature and introducing the whole organ into a recipient so that the organ is supplied with blood by vasculature belonging to the recipient. Further disclosed is a method of preserving fertility of a patient undergoing a treatment expected to cause sterility. The method include removing a whole gonadal organ from the patient, cryo-preserving the whole gonadal organ,conducting the treatment and waiting for an effect thereof to subside, thawing the whole gonadal organ and introducing the whole gonadal organ where it is supplied with blood by the vasculature system of the patient. Further disclosed is a method of assuring a supply of viable gametes for future use, the method includes removing a whole gonadal organ from a patient, cryo-preserving the whole gonadal organ, waiting until viable gametes are desired by the patient, thawing the organ and introducing the gonadal organ into the patient so that it is supplied with blood by a vasculature system belonging to the patient.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to methods of preserving functionality ofan organ, preserving fertility of a patient undergoing a treatmentexpected to cause sterility and assuring a supply of viable gametes forfuture use and, more particularly, to a methods which rely upon removalof an organ, cryopreservation of the removed organ and reintroduction ofthe cryopreserved organ into a recipient in such a way that thevasculature of the recipient supplies blood to the introduced organ or aportion thereof.

Undoubtedly, successful cryopreservation of solid organs of clinicalinterest would have a significant impact on the field of organtransplantation. However, despite several decades of research freezingand thawing of solid organs remains largely impractical (Dietzman etal., 1973; Guttman et al., 1977; Pegg et al., 1987; Karow, 1981;Jacobsen et al., 1982).

Attempts to understand the mechanism by which cold-blooded animalssurvive freezing in nature (Storey and Storey, 1988; Storey, 1990) haveled to successful, short-term cryopreservation of hearts and livers byfreezing at high subzero temperatures (−3.4° C. to −4° C.), (Wang etal., 1992; Banker et al.,1991, Rubinsky et al., 1994, Soltys et al.,2001).

Cryopreservation at lower temperatures, which is necessary for long-termstorage, was attempted in dog intestines, but vascular injury wasobserved after thawing (Karlasson et al., 1996). Livers have regainedpartial function after freezing to −60° C. (Mazur, 1977), dog spleens(Fuller, 1987) and ureters (Pegg, 1987) have survived deep freezing andtransplantation, and kidneys have survived freezing to −80° C.

(Fahy et al., 1984) proposed vitrification (ice free cryopreservation)as an alternate strategy for for organ cryopresernation (Kheirabadi etal., 2000). Vitrification produced a high survival rate in cryopreservedsmall organisms such as Drosophila embryos at −196° C. (Steponkus etal., 1990; Mazur et al., 1992).

Thus, successful cryopreservation of organs apparently requires aspecific optimal cooling rate, because damage may occur if organs arefrozen either too rapidly or too slowly (Karlsson and Toner., 1996;Mazur, 1977). Cooling too slowly (<1° C./min) causes extracellularcrystallization which may physically disrupt the vital tissuearchitecture (Fuller, 1987; Pegg, 1987), whereas at higher cooling ratesintracellular crystallization will cause irreversible damage.

Further, cryopreservation of large-volume samples, such as tissues ororgans, introduces heat transfer problems. In macroscopic samples thereis a large thermal gradient from the surface of the sample to theinterior. For example, it was shown that cells survived equally whenfrozen as isolated cells or in a monolayer, only when the appliedcooling rate was less than 0.5° C./min. Moreover, the survival of cellsin monolyer was higher than isolated cells using the determined optimalcooling rate of 0.2° C./min. This indicates that the attached cells weremore tolerant of slow cooling injury (Armitage et al., 1996). The needfor a slow cooling rate is further illustrated by the cooling rate atwhich a Wood frog survives freezing (less than 0.1° C./minute; Schmid,1982).

Typically, prior art freezing techniques produce temperature gradientswithin the freezing chamber which make it difficult to achieve anoptimal cooling rate (Koebe et al., 1993). In addition, cooling ratesslower than 0.1° C./minutes are hard to control in a programmablefreezing apparatus since the accuracy of the temperature measurement iswithin that range (OMEGA ENGINEERING, INC). U.S. Pat. No. 5,873,254 toArav teaches a device capable of producing a uniform cooling rate of0.1° C./min throughout a bilogical sample. However, the earlierteachings of Arav do not disclose methods for cryopreservation of wholeorgans and subsequent introduction of those whole organs into arecipient subject. Specifically, the earlier teachings of Arad do notinclude methods for thawing a whole organ without impairingfunctionality thereof and surgical techniques for anastomictransplantation.

It is well established that treatment of a malignant disease byradiotherapy or chemotherapy can have dramatic and irreversible effect,on fertility, especxially in female patients (Byrne et al., 1987; Ataya,1989; Familiari, 1993).

Thus, there is a recognized need, but no established method, forcryopreservation of a gonadal organ for subsequent reimplantation forpatients at risk for premature sterility as a result of planned cancertreatment. While oocyte cryopreservation theoretically offers a means ofpreserving fertility for these patients, severe practical problems foroocyte cryopreservation remain unsolved (Arav et al., 1996; Zeron etal., 1999; Zenzes et al., 2001). Similarly, while cryopreservation ofsemen for male patients is available, it is not an optimum method ofassuring male sterility. Cryopreservation of oocytes and/or sementypically require laboratory intervention in the form of IVF procedures.This prospect is daunting to many patients for a variety of reasonsincluding emotional and religous reasons. Thus, storage and later use ofof a gonadal organ for conception by conventional methods offerssignificant advantages over previously available alternatives.

A high proportion of viable follicles have been found to survive inhuman ovarian tissue after freeze-thawing (Hovatta et al., 1996; Newtonet al.,1996; Oktay et al., 1997), and this has aroused interest in theprocedure as a potential means of preserving the fecundity of patientsat risk of premature ovarian failure (Donnez et al., 1998; Newton etal., 1998). However, freezing and thawing of the whole ovary was notreported.

Gosden and colleagues (1994) have achieved limited success in sheepusing ovarian cortex freezing leading to speculation that this techniquemay be applicable to humans. The sheep ovary is similar to the humanovary in that it has a dense fibrous stroma and relatively high densityof primordial follicles in the ovarian cortex.

Autotransplantation of frozen-banked and fresh ovarian cortexcryopreservation ovarian cortex, have resulted in two pregnancies(Gosden et al., 1994). Baird et al. (1999) performed frozen tissueautotransplantation in eight sheep, which were monitored for up to 22months. All the animals resumed cyclicity and showed hormone production,however, it was established that there was a drastic reduction in thetotal follicular number, and the resumption of cyclical ovarian functionwas temporary. Thus, despite recent advances in this area;transplantation of a functional portion of an ovary for purposes ofrestoring full ovarian function remains unreliable. Therefore, freezingand grafting of human ovarian tissue is not considered clinical option(Kim et al., 2001).

Clinical acceptability of ovarian tissue transplantation will requirehigher numbers of follicles survive and retain the capability to developand ovulate. Recently it has been demonstrated that during freezing ofcortical ovarian slices granulosa cells are more damaged than oocytes(Siebzehnrubl et al., 2000).

Further, currently accepted experimentyal practice idictates use ofovarian slices resulting in schemic damage as a rersult of non-vasculartransplantation. Still further, the need for an IVF procedure inaddition to preparing tissue for cryopreservation is time consuming andincreases costs of the procedure. Finally, in those cases where ovarianfunction has been restored, long post=implantation delays are observed(Radford et al., 2001).

Transplantation of whole ovary including vascular has been recognoziedas a theoretical method of solving the problems described above. WhileOvary transplantation has been known for decades, (Nobel Lecture, Dec.11, 1912:“ Suture of blood-vessels and transplantation of organs”),autotransplantation has been considered impractical because of theabsence of long term organ cryopreservation.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, methods of preserving functionality of an organ,methods of preserving fertility of a patient undergoing a treatmentexpected to cause sterility and methods of assuring a supply of viablegametes for future use devoid of the above limitation.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of preserving functionality of an organ. The method includes: (a)removing at least a whole organ including vasculature from a donor; (b)cryo-preserving the whole organ including vasculature; (c) allowing aperiod of time to elapse; (d) thawing the whole organ includingvasculature so that at least a portion of cells contained therein resumebiological activity; and (e) introducing the whole organ includingvasculature into a recipient so that a blood vessel present in thevasculature of the whole organ including vasculature is supplied withblood by a vasculature system belonging to the recipient. For purposesof this specification and the accompanying claims, the phrase “wholeorgan including vasculature” refers to those blood vessels responsiblefor local supply of blood to the removed organ.

According to another aspect of the present invention there is provided amethod of preserving fertility of a patient undergoing a treatmentexpected to cause sterility, the method includes; (a) removing at leastone whole gonadal organ including vasculature from the patient; (b)cryo-preserving the at least one whole gonadal organ includingvasculature; (c) conducting the treatment and waiting for an effectthereof to subside; (d) thawing the at least one whole gonadal organincluding vasculature so that at least a portion of cells containedtherein resume biological activity; and (e) introducing at least onewhole gonadal organ including vasculature into the patient so that ablood vessel present in the gonadal organ is supplied with blood by avasculature system belonging to the patient.

According to yet another aspect of the present invention there isprovided a method of assuring a supply of viable gametes for future use.The method includes;(a) removing at least one whole gonadal organincluding vasculature from a patient; (b) cryo-preserving the at leastone whole gonadal organ including vasculature; (c) waiting until viablegametes are desired by the patient; (d) thawing the gonadal organ sothat at least a portion of cells contained therein resume biologicalactivity; and (e) introducing the at least one whole gonadal organincluding vasculature the patient so that the at least one whole gonadalorgan including vasculature is supplied with blood by a vasculaturesystem belonging to the recipient.

According to further features in preferred embodiments of the inventiondescribed below, a single individual serves as both the donor and therecipient.

According to still further features in the described preferredembodiments the whole organ including vasculature has a capacity tofulfill at least one function selected from the group consisting ofoogenesis, spermatogenesis, supply of a hormone, supply of a growthfactor, metabolism of a precursor into an end-product, elimination ofwaste and regulation of a physiologic process.

According to still further features in the described preferredembodiments the method further includes facilitating angiogenesis of thewhole organ including vasculature.

According to still further features in the described preferredembodiments the organ is a gonadal organ selected from the groupconsisting of an ovary and a testicle.

According to still further features in the described preferredembodiments the period of time is selected from the group consisting of:(i) sufficient time for resolution of an acute medical condition of thedonor; (ii) sufficient time for a change in a physiologic state of therecipient; (iii) sufficient time for identifying a recipient unlikely toreject tissue from the donor.

According to still further features in the described preferredembodiments the single individual is a cancer patient attempting toavoid sterility.

According to still further features in the described preferredembodiments the single individual is a woman wishing to alleviatemenopausal symptoms by implantation of a previously removed ovaryincluding vasculature after onset of menopause.

According to still further features in the described preferredembodiments the single individual is a woman wishing to achieve alteredfertility status by implantation of a previously removed whole ovaryincluding vasculature.

According to still further features in the described preferred the atleast one whole gonadal organ including vasculature is selected from thegroup consisting of an ovary and a testicle.

According to still further features in the described preferredembodiments the method of further includes installing at least oneprosthetic testicle in a scrotum of the patient.

According to still further features in the described preferredembodiments the method of further includes administering sex hormones tothe patient.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing methods of preservingfunctionality of an organ, preserving fertility of a patient undergoinga treatment expected to cause sterility and assuring a supply of viablegametes for future use.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a cartoon of a device used for freezing of a whole organsuitable for use in the context of the present invention;

FIG. 2 is a flow diagram illustrating essential steps in methodsaccording to the present invention;

FIGS. 3 a-d are photomicrographs of oocytes (panels a and b) and enbryos(panels c and d) from experimental (panels a and c) and control (panelsb and d) ovaries; and

FIG. 4 is a graph illustrating estradiol and progesterone levels as afunction of time post transplantation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods of preserving functionality of anorgan, preserving fertility of a patient undergoing a treatment expectedto cause sterility and assuring a supply of viable gametes for futureuse which can be used to remove an organ, cryopreserve the removed organand reintrodue the cryopreserved organ into a recipient.

Specifically, the present invention assures that the vasculature of therecipient supplies blood to the introduced organ or a portion thereof.

For purposes of better understanding the present invention, asillustrated in Figures XXX of the drawings, reference is first made tothe construction and operation of a conventional (i.e., prior art)thermal gradient device as illustrated in FIG. 1.

A freezing device 20 (Multi-Gradient-Directional (MGD) IMT technologies;Israel) according to earlier teachings of Arav (U.S. Pat. No. 5,873,254)was employed to freeze removed whole organs. In brief, device 20includes a plurality of temperature domains (e.g. 22, 24, 26 and 28)within 270 mm copper blocks 36. A tube containing a whole organ 30 ispushed at a constant velocity (V; 32) through the predeterminedtemperature gradient (34; G=ΔT/d where ΔT is temperature differences andd is the distance between temperatures) resulting in a cooling rate (B)according to the equation B=G*V. The cooling rate may be st to, forexample, 0.03° C./min by varying velocity 32 at which tube 30 containingthe organ passes through the gradient 34. Seeding is typically performedat the tip of the tube 30 and ice interphase is propagated according tothe freezing point of the solution.

The principles and operation of methods according to the presentinvention may be better understood with reference to the drawings andaccompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Referring now to the drawings, FIG. 2 illustrates a method 40 ofpreserving functionality of an organ. Method 40 includes removing 42 atleast a whole organ including vasculature from a donor. Method 40further includes cryo-preserving 44 the whole organ includingvasculature and allowing 46 a period of time to elapse. Method 40further includes thawing 48 the whole organ including vasculature sothat at least a portion of cells contained therein resume biologicalactivity. Method 40 further includes introducing 50 the whole organincluding vasculature into a recipient so that a blood vessel present inthe vasculature of the whole organ including vasculature is suppliedwith blood by a vasculature system belonging to the recipient.Preferably, a single individual serves as both the donor and therecipient. The single individual may be, for example, a cancer patientattempting to avoid sterility. Alternately, the single individual may bea woman wishing to alleviate menopausal symptoms by implantation of apreviously removed ovary including vasculature after onset of menopause.Alternately, but also preferably, the single individual is a womanwishing to achieve altered fertility status by implantation of a atleast one previously removed whole ovary including vasculature. In thiscase, a woman may elect to have one or both ovaries remove when she isrelatively young, and then have one or both ovaries re-implanted at alater time when she wishes to conceive.

According to various preferred embodiments of method 40, the whole organincluding vasculature may be introduced to fulfill at least one functionincluding, but not limited to, oogenesis, spermatogenesis, supply of ahormone, supply of a growth factor, metabolism of a precursor into anend-product, elimination of waste or regulation of a physiologicprocess. Most preferably, the organ is a gonadal organ i.e. an ovary ora testicle.

Preferably, method 40 further includes facilitating angiogenesis of thewhole organ including vasculature.

Allowing a period of time to elapse may be for a variety of reasons,such as, for example, allowing sufficient time for resolution 52 of anacute medical condition of the donor, or allowing sufficient time for achange 54 in a physiologic state of the recipient, or allowingsufficient time for identifying 56 a recipient unlikely to reject tissuefrom the donor.

The invention is further embodied by method 40 of preserving fertilityof a patient undergoing a treatment expected to cause sterility. Method40 includes removing 42 at least one whole gonadal organ includingvasculature from the patient. Method 40 further includes cryo-preserving44 the at least one whole gonadal organ including vasculature. Method 40further includes conducting 45 the treatment and waiting for an effectthereof to subside. Method 40 further includes thawing 48 the at leastone whole gonadal organ including vasculature so that at least a portionof cells contained therein resume biological activity; and introducing50 the at least one whole gonadal organ including vasculature into thepatient so that a blood vessel present in the gonadal organ is suppliedwith blood by a vasculature system belonging to the patient. The atleast one whole gonadal organ including vasculature is either an ovaryor a testicle depending upon the sex of the patient.

In cases where the gonadal organ is a testicle, method 40 preferablyfurther includes installing 58 at least one prosthetic testicle in ascrotum of the patient. This step is performed to prevent damage to thepatient's self image.

Whether the patient is male or female, method 40 further includesadministering 60 sex hormones to the patient if both gonadal organs areremoved so that secondary sex characteristics are not affected.

The present invention is further embodied by method 40 of assuring asupply of viable gametes for future use. Method 40 includes removing 42at least one whole gonadal organ including vasculature from a patient.Cryo-preserving 44 the at least one whole gonadal organ includingvasculature and waiting until viable gametes are desired 47 by thepatient. Method 40 further includes thawing 48 the gonadal organ so thatat least a portion of cells contained therein resume biological activityand introducing 50 the at least one whole gonadal organ includingvasculature into the patient so that the at least one whole gonadalorgan including vasculature is supplied with blood by a vasculaturesystem belonging to the recipient.

Freezing of whole organs in conjunction with methods 40 may beaccomplished, for example, using devices taught by Arav (U.S. Pat. No.5,873,254) for example by placing the whole organ in a volume offreezing solution; pushing the solid tissue at a velocity (V) through apredetermined temperature gradient (G) thus determining a cooling rate(B) according to the equation B=G*V; initiating crystallization at apredetermined position within the temperature gradient (G); and allowingthe solid tissue to continue to move through the temperature gradient(G) at the velocity. According to Arav G=ΔT/d where ΔT is thetemperature difference and d is the distance over which it ismaintained. Thawing of the cryo=preserved organ may be accomplished, forexample, by placing the cryopreserved organ into a 66° C. environmentfor a first period of tim and placing the cryopreserved solid tissueinto a 38° C. water bath for a second period of time as describedhereinabove.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical, andcell biology techniques. Such techniques are thoroughly explained in theliterature. See, for example, “Molecular Cloning: A laboratory Manual”Sambrook et al., (1989); “Current Protocols in Molecular Biology”Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “CurrentProtocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md.(1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley &Sons, New York (1988); Watson et al., “Recombinant DNA”, ScientificAmerican Books, New York; Birren et al. (eds) “Genome Analysis: ALaboratory Manual Series”, Vols. 1-4, Cold Spring Harbor LaboratoryPress, New York (1998); methodologies as set forth in U.S. Pat. Nos.4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology:A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994);“Culture of Animal Cells—A Manual of Basic Technique” by Freshney,Wiley-Liss, N.Y. (1994), Third Edition; “Animal Cell Culture” Freshney.R. I., ed. (1986); “Methods in Enzymology” Vol. 1-317, Academic Press;“PCR Protocols: A Guide To Methods And Applications”, Academic Press,San Diego, Calif. (1990);); all of which are incorpotaed by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Materials and Methods

Reference is first made to the following materials and methods which aregenerally employed in performing experiments described in examples setforth hereinbelow.

Cryopreservation of organs. Organs (e.g. ovaries) were removed fromdonor animals and perfused with Belzer UW solution(ViaSpan®, Do PontPharma, USA) +10% dimethyl sulfoxide( DMSO; Sigma Chemicals, St. Louis,USA) by insertion of a catheter into a blood vessel of the organ.

Organs, and their vasculature network, were then transferred to 16×100mm glass tubes (Manara, Israel) and frozen using a commerciallyavailable (IMT, Israel) multi-gradient directional freezing apparatus(described in U.S. Pat. No. 5,873,254). Different freezing temperatureswere assayed as described hereinbelow.

Cryo-preservation apparatus. A Cryopreservation apparatus as describedby Arav (U.S. Pat. No. 5,873,254; FIG. 1) was employed for freezing theovaries as described hereinabove. Briefly, the apparatus includes 4temperature domains within 270 mm copper blocks. A test tube containingthe sample is pushed at a constant velocity (V) through thepredetermined temperature gradient (G=ΔT/d where ΔT is temperaturedifferences and d is the distance between temperatures) resulting in acooling rate (B) according to the equation B=G*V. The cooling rate wasset to 0.1° C./min by varying the speed the tube passes through thetemperature gradient. Seeding was performed at the tip of the test tubeand ice interphase was propagated according to the freezing point of thesolution.

In-Vitro Follicular Assays:

Follicles were aspirated using a 22 gauge needle connected to a 5 mlsyringe and thin ovarian cortex slices were cut. The granulose cells andfollicular viability was assessed using FDA (fluorescein diacetate)/DAPI(4,6-diainidino-2-phenylindole) fluorescent live/dead staining(Molecular Probes, Leiden, the Netherlands and Merk, Germany).

Thawing of Cryopreserved Organs:

Organs were first immersed in a high temperature (66-68 ° C.) water bathfor 10 to 20 seconds followed by immersion in a 38-40° C. water bath for10 to 120 seconds. Ovaries were then perfused with 0.5M sucrose and 150iu heparin to remove cryoprotectants and prevent blood clotting.

Introduction of Organs into Recipients:

Transplantation was performed by re-anastomizing arteries and veins ofthe transplanted organ with 10 zero sutures (Ethilon; Johnson andJohnson, U.S.A.). Success of the procedure was evaluated by observingarterial blood pulse and venous return flow in the newly introducedorgan. A hyaluronic acid gel was employed to prevent adhesions(Intergel; Johnson and Johnson, U.S.A.). For ovary transplantation wasautologus.

Fertility Evaluation of Transplanted Ovaries:

Two months after transplantation, 600 iu of PMSG were administered tothe recipient to cause superovulation (Intervet International BV;Boxmeer, Netherlands). Three days later follicles were aspirated fromthe transplanted organ using a syringe and a 20 gauge needle. Oocyteswere transported in a mobile incubator (Minitube, Germany) at 38° C. inbuffer medium to an IVF laboratory. Aspirated oocytes were then put intoTCM-199 maturation medium supplemented with 10% (V/V) heat-inactivatedfetal calf serum (FCS) (Bio-lab, Jerusalem, Israel), 0.2 mmol Napyruvate l⁻¹, 5 μg gentamicin l⁻¹, 10 μg ovine LH ml⁻¹ (NIADDK-NIH-26,AFP5551B, Bethesda, Md.), 1 μg ovine FSH ml⁻¹ (NIADDK-NIH-20, AFP7028D,Bethesda, Md.) and 1 μg estradiol ml⁻¹ and incubated in a 38.5° C. in ahumidified atmosphere of 5% CO₂ in air. After maturation, oocytes weredenuded from cumulus cells in the maturation wells and placed for 5 min.in the ionomycin medium: 10 ml TCM-199, supplemented with 10% (V/V)heat-inactivated FCS, 0.2 mmol Na pyruvate l⁻¹, 5 μg gentamicin l⁻¹, and5 μmol of ionomycin l⁻¹. Oocytes were transferred to6-dimethylaminopurine (6-DMAP) medium (10 ml of TCM 199 supplementedwith 2 mmol of 6-DMAP l⁻¹) for 4.5 h. The oocytes were then washed threetimes in a cleavage medium (Sydney IVF Cleavage Medium, Cook, Australia)and transferred into 50 μl cleavage drops under mineral oil for anotherthree days. Cleaved embryos were counted on day 4 (activation day=0) andtransferred to 50 μl drops under mineral oil of blastocysts medium(Sydney IVF Blastocyst Medium, Cook, Australia). Blastocysts werecounted after 8-10 days.

Hormonal status. Progesterone and estradiol levels were analyzed inserum samples bi-weekly. Progesterone was measured using COAT-A-COUNTProgesterone DPC®,USA. Estradiol levels were measured by ESTER-US-CT,ORIS Group, France.

EXAMPLE 1 In Vitro Assay of Viability of Sheep Ovaries After Thawing

In order to determine the effect of freezing temperature on subsequentviability, fifteen Assaf sheep ovaries were collected 10 min afterslaughter. The ovaries were perfused with a Belzer UW solution(ViaSpan®,Do Pont Pharma, USA)+10% DMSO (Sigma, St. Lois, USA) for 3 min, in orderto obtain maximal permeability of the organ to DMSO. After perfusion,the ovaries were loaded into 16×100 mm glass test tubes (Manara, Israel)in UW+10% DMSO and transferred to the freezing apparatus (IMT, Israel)described hereinabove. Control ovaries were perfused as above but werenot cryopreserved. Ovaries were frozen to different temperatures asdetailed in table 1. TABLE 1 Freezing parameters of whole ovine ovariesEXP. T1 T2 T3 T4 T5 V CR 1 0° C. −6° C. −10° C. −14° C. — 0.03 0.1° mm/sC./min 2 0° C. −6° C. −18° C. −30° C. — 0.01 0.1° mm/s C./min 3 0° C.−6° C. −18° C. −30° C. −196° C. 0.01 0.1° mm/s C./min

Briefly, the cooling rate was set to 0.1° C./min by varying the speed ofthe tube through the temperature gradient. Seeding was performed at thetip of the test tube and ice interface was propagated according to thefreezing point of the solution. 15 minutes after reaching the finaltemperature the test tube was thawed. When −196° C. was used as thefinal temperature, ovaries were stored in LN for 2 days to one weekbefore being thawed.

Frozen ovaries were thawed as described hereinabove. The frozen-thawedwhole ovaries were then washed in buffer medium at 38° C. Follicles wereaspirated with a 22 G needle connected to a 5 ml syringe in order toobtain granulosa cells. Thin slices were cut from the ovarian cortex.The granulose cells and primordial and small antral follicules wereevaluated for viability using fluorescent microscopy as described byOktay et al. (Fertil. and Steril. 1997(67):481-486) FDA (fluoresceindiacetate)/DAPI (4,6-diamidino-2-phenylindole) fluorescent live/deadstaining (Molecular Probes, Leiden, the Netherlands and Merk, Germany)was performed.

Granulosa cells that were evaluated after freezing and thawing showed nostatistically significant differences from controls (Table 2). Thesurvival of small follicles (small antral and primordial follicules)showed no statistically significant differences from controls (Table 2).Means were calculated using the General Linear Model procedure of JMP(SAS Institute, 1994) and differences between treatments examined byanalysis of variance. Significance was P<0.05.

These results clearly demonstrate that small follicles and granulosecells are not damaged by freezing and thawing using conditions describedherein. TABLE 2 In vitro study survival of ovaries after freezing todifferent temperatures Number Viability of of Storage granulosa Livefollicles/ Exp. ovaries temp. cells(±SE) dead follicles Cont. 3 Room 50%± 7  101/105 temp. 1 3  −14° C. 38% ± 29 69/69 2 3  −30° C. 40% ± 1618/37 3 5 −196° C.    58 ± 16 68/73

EXAMPLE 2 In Vivo Assay of Viability Sheep Ovaries After Thawing

In order to verify the results of Example 1 in an in vivo system,eleven-month-old Assaf sheep were subjected to oophorectomy undergeneral anaesthesia via longitudinal laparotomy. The uterus and ovarieswere exposed and a dissection of the right ovarian artery and vein wasperformed. The ovary was perfused in vivo with UW+10% DMSO for 3 min. Itwas then excised and inserted into a freezing tube containing UW+10%DMSO. Slow freezing was performed by cooling from 0 to −6° C. over 2min, seeding was done before entering −6° C. and cooling to −14° C.,−30° C. and thawing after 15 minutes or to −30° C. and then directplunging into LN and then thawing. The cooling rate of the threedifferent procedures was 0.1° C./min. After 15 min at the finaltemperature thawing was performed by plunging the test tube into a 66°C. water bath for 20 s and then into a 40° C. water bath for 2 min.Ovarian vascular transplantation was performed by re-anastomosing theovarian artery and vein with 10 zero sutures (Ethilon, Johnson andJohnson). After the arterial anastomosis was completed, blood flow wasverified by observing blood pulse in the ovary and venous return throughthe ovarian vein.

Two months after autotransplantation, 600 iu PMSG (intervetinternational BV, Boxmeer-Holand, France: intervet SA 49100-Angers,Pais-Bas) was administered. Oocytes were aspirated three days later,matured, and parthanogenetically activated in vitro as describedhereinabove.

Parthenogenetic activation resulted in normal development of ⅗ retrievedoocytes., pictures of oocytes (FIGS. 3 a and b) and embryos (FIG. 3 cand) indicate no apparent fifference between experimental (FIGS. 3 a andc) and control (FIGS. 3 b and d) oocytes or embryos. Followingoophorectomy of the unfrozen contralateral ovary in the recipient, bloodwas taken bi-weekly for two months and progesterone and estradiol levelswere evaluated using a commercially available assay (Coat-A-CountProgesterone, DPC, LA, USA and ESTR-US-CT, ORIS Group, France) Resultsare summarized in FIG. 4.

Briefly, estrogen and progesterone levels dropped to negligible levelsimmediately post transplantation. Estrogen levels surged four weeksfollowing transplantation of an ovary frozen in liquid nitrogen.Cyclical estrogen and progesterone thereafter were measured for overthirty weeks. These results clearly demonstrate that the implanted ovaryrestored normal hormonal balance to the recipient. Further, normaloocyte division and development (FIG. 3) indicate that the primordialand small antral follicles in the transplanted ovary recover fromfreezing without ill effect. In summary, these results indicate that itis now feasible to remove and freeze a solid organ for subsequenttransplantation into a recipient and to achieve full physiologicactivity from the thawed transplanted organ. This capacity is expectedto find utility in a variety of clinical contexts as detailkedhereinabove.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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1. A method of preserving functionality of an organ, the methodcomprising: (a) removing at least a whole organ including vasculaturefrom a donor; (b) crylo-preserving said whole organ includingvasculature; (c) allowing a period of time to elapse; (d) thawing saidwhole organ including vasculature so that at least a portion of cellscontained therein resume biological activity; and (e) introducing saidwhole organ including vasculature into a recipient so that a bloodvessel present in said vasculature of said whole organ includingvasculature is supplied with blood by a vasculature system belonging tosaid recipient.
 2. The method of claim 1, wherein a single individualserves as both said donor and said recipient.
 3. The method of claim 1,wherein said whole organ including vasculature has a capacity to fulfillat least one function selected from the group consisting of oogenesis,spermatogenesis, supply of a hormnone, supply of a growth factor,metabolism of a precursor into an end-product, elimination of waste andregulation of a physiologic process.
 4. The method of claim 1, furtherincluding facilitating angiogenesis of said whole organ includingvasculature.
 5. The method of claim 1, wherein the organ is a gonadalorgan selected from the group consisting of an ovary and a testicle. 6.The method of claim 1, wherein said period of time is selected from thegroup consisting of: (i) sufficient time for resolution of an acutemedical condition of said donor; (ii) sufficient time for a change in aphysiologic state of said recipient; (iii) sufficient time foridentifying a recipient unlikely to reject tissue from said donor. 7.The method of claim 2, wherein said single individual is a cancerpatient attempting to avoid sterility.
 8. The method of claim 2, whereinsaid single individual is a woman wishing to alleviate menopausalsymptoms by implantation of a previously removed ovary includingvasculature after onset of menopause.
 9. The method of claim 2, whereinsaid single individual is a woman wishing to achieve altered fertilitystatus by implantation of a previously removed whole ovary includingvasculature.
 10. A method of preserving fertility of a patientundergoing a treatment expected to cause sterility, the methodcomprising; (a) removing at least one whole gonadal organ includingvasculature from the patient; (b) cryo-preserving said at least onewhole gonadal organ including vasculature; (c) conducting the treatmentand waiting for an effect thereof to subside; (d) thawing said at leastone whole gonadal organ including vasculature so that at least a portionof cells contained therein resume biological activity; and (e)introducing at least one whole gonadal organ including vasculature intothe patient so that a blood vessel present in said at least one gonadalorgan is supplied with blood by a vasculature system belonging to thepatient.
 11. The method of claim 10 wherein said at least one wholegonadal organ including vasculature is selected from the groupconsisting of an ovary and a testicle.
 12. The method of claim 10,further comprising installing at least one prosthetic testicle in ascrotum of the patient.
 13. The method of claim 10 further comprisingadministering sex hormones to the patient.
 14. A method of assuring asupply of viable gametes for future use, the method comprising; (a)removing at least one whole gonadal organ including vasculature from apatient; (b) cryo-preserving said at least one whole gonadal organincluding vasculature; (c) waiting until viable gametes are desired bythe patient; (d) thawing said gonadal organ so that at least a portionof cells contained therein resume biological activity; and (e)introducing said at least one whole gonadal organ including vasculaturethe patient. so that said at least one whole gonadal organ includingvasculature is supplied with blood by a vasculature system belonging tothe patient.
 15. The method of claim 14 wherein said gonadal organ isselected from the group consisting of an ovary and a testicle.
 16. Themethod of claim 14, further comprising installing at least oneprosthetic testicle in a scrotum of said patient.
 17. The method ofclaim 14 further comprising administering sex hormones to said patient.